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Results for “Microinjection”

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PubMed2001

Inhibitory effect of aluminium on the axonal transport of HRP microinjected into dorsal root ganglion neurons in vitro.

In the present study the function of axonal transport in individual neurons under aluminium intoxication was investigated experimentally in comparison with controls. We used the technique of microinjection of horseradish peroxidase (HRP) in dissociated dorsal root ganglia (DRG) neurons and neurons of explant cultures of DRG. Different exposure periods (1 and 6 hours as well as 6 and 10 days) to aluminium were analysed quantitatively. This analysis revealed an impaired anterograde transport of HRP already after a short aluminium intoxication period of only 1 hour in DRG cells in vitro, an effect that increased with a prolonged aluminium exposure for up to 10 days. Hence, functional alterations of the anterograde transport caused by aluminium could be detected even after short exposure periods. Furthermore, the effects of aluminium on anterograde transport mechanisms were reversible 8 days after removal of aluminium. To determine how aluminium affects the cytoskeleton, we performed immunohistochemistry and electron microscopy on cultured DRG neurons. Distinct morphological alterations of the cytoskeleton, especially the accumulation of phosphorylated neurofilaments, appeared after 6 days of aluminium exposure. Our results suggest that neurofilaments are indispensable to the functional integrity of the cytoskeleton and its ability to mediate microtubule-based axonal transport processes.

Aluminum↗

PubMed1988

Effects of opioid microinjections into the medial septal area on spatial memory in rats.

Recent work has demonstrated that posttraining systemic opioid antagonist administration facilitates the acquisition of a radial arm maze task in new spatial environments. In this study, we examined the effect of posttraining naloxone and beta-endorphin microinjections into the medial septal area on the acquisition of a radial maze task in new spatial environments. The results of these experiments demonstrated that posttraining intraseptal naloxone administration facilitated, whereas posttraining intraseptal beta-endorphin administration impaired, the acquisition of criterion performance on a maze task performed in new spatial environments. Further, intraventricular beta-endorphin administration did not produce effects that were comparable to those observed following intraseptal beta-endorphin administration, which indicates that the septal region is a brain site that is sensitive to the effects of opioids on spatial memory in new environments. Further, posttraining intraseptal beta-endorphin administration had no effect on working memory in a familiar spatial environment, whereas pretraining intraseptal beta-endorphin administration had no effect on the performance of a previously acquired spatial task.

Animals↗

PubMed1994

Chlordiazepoxide microinjected into the region of the dorsal raphe nucleus eliminates the interference with escape responding produced by inescapable shock whether administered before inescapable shock or escape testing.

Systemic administration of benzodiazepines before exposure to inescapable shock (IS) blocks the enhanced fear conditioning and escape learning deficits that follow exposure to IS, whereas administration before the subsequent behavioral testing eliminates the enhanced fear but not the interference with escape. The failure of benzodiazepines to reduce the IS-produced escape learning deficit when given before testing is inconsistent with a recent proposal that interference with escape is mediated by an IS-induced sensitization of dorsal raphe nucleus (DRN) activity. The present experiments demonstrate that chlordiazepoxide will block both the enhancement of fear and interference with escape responding when given before either IS or testing if microinjected in the region of the DRN. This suggests that systemic benzodiazepines fail to block escape deficits when given before testing because action at a site distant from the DRN counters the effect of the drug at the DRN.

Animals↗

PubMed1995

8-OH-DPAT microinjected in the region of the dorsal raphe nucleus blocks and reverses the enhancement of fear conditioning and interference with escape produced by exposure to inescapable shock.

Prior work suggests that inhibition of the dorsal raphe nucleus (DRN) either during exposure to inescapable electric shock (IS) or during later behavioral testing might block the usual behavioral consequences of IS. The 5-HT1A agonist 8-OH-DPAT was microinjected into the region of the DRN either before exposure to IS or before testing for fear conditioning and escape learning conducted 24 hr later. IS potentiated fear conditioning and interfered with escape performance. These effects were completely prevented by intra-DRN administration of 8-OH-DPAT at either point. Low but not high systemic doses of 8-OH-DPAT had a similar effect, supporting the idea that the effective site of action is presynaptic. The relation between these data and other effects of 8-OH-DPAT is discussed.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1995

Increases in vocalization and motor reflex thresholds are influenced by the site of morphine microinjection: comparisons following administration into the periaqueductal gray, ventral medulla, and spinal subarachnoid space.

The relative influence of morphine microinjected into the periaqueductal gray, ventral medulla (nucleus raphé magnus or nucleus reticularis gigantocellularis), or spinal subarachnoid space on the thresholds of responses organized at spinal (spinal motor reflexes, SMRs), medullary (vocalizations elicited during shock, VDSs), and rhinencephalic-diencephalic (vocalization after discharges, VADs) levels of the neuraxis was assessed. Dose-dependent increases in response thresholds differed with the site of morphine injection. These results indicate that the mu-opiate-receptor-linked systems in the mesencephalon, medulla, and spinal cord exert differential antinociceptive effects on pain behaviors organized at different levels of the neuraxis. A hypothesis is offered regarding the mechanisms through which morphine inhibits nociceptive transmission through various levels of the CNS. VADs are promoted as a model system for analyzing the affective-motivational dimension of the pain experience.

Affect↗

PubMed1996

The differential contribution of spinopetal projections to increases in vocalization and motor reflex thresholds generated by the microinjection of morphine into the periaqueductal gray.

The capacity of morphine microinjected into the ventrolateral periaqueductal gray (vPAG) to elevate the thresholds of spinal motor reflexes (SMRs), vocalizations during shock (VDSs) and vocalization afterdischarges (VADs) was challenged by the intrathecal administration of receptor antagonists to serotonin (methysergide), norepinephrine (phentolamine) and mu-opiates (naloxone). Methysergide and phentolamine were equipotent in reversing increases in SMR thresholds. The efficacy of these antagonists to reduce increases in VDS and VAD thresholds was dependent on the dose of morphine administered into the vPAG. These results indicate that the dose of morphine administered into the vPAG determines the contribution of spinopetal projections in inhibiting dorsal horn neurons involved in reflex generation versus the rostral transmission of pain information. A hypothesis is offered regarding the mechanisms by which vPAG administered morphine suppresses nociceptive transmission through different levels of the neuraxis.

Animals↗

PubMed1997

The development of a conditioned place preference to morphine: effects of microinjections into various CNS sites.

Experiment 1 examined whether microinjections of morphine (1 microg in 0.5 microl over 1 min x 2 pairings) into 13 different CNS sites produced a conditioned place preference (CPP). Injections into the lateral ventricles (LV), ventral tegmental area (VTA), or periaqueductal gray (PAG) produced a CPP; injections 1 mm dorsal to the PAG or VTA, or into the caudate putamen, medial frontal cortex, hippocampus, lateral nucleus of the amygdala, lateral hypothalamus, pedunculopontine tegmental nucleus, posterior hypothalamus, ventral palladium, or nucleus accumbens septi (core or shell) did not. In Experiment 2, morphine 0.2 microg produced a CPP when injected into the VTA but not in the PAG, while 5.0 microg was effective in both sites. The CPP induced by systemic morphine (4 mg/kg x 1 pairing) was blocked by naloxone methiodide (NM) injected (2 nmol in 0.5 microl) into the VTA. PAG injections of 2 nmol reduced, and 5 nmol NM eliminated, the CPP. The results confirm that morphine injections into the VTA or the PAG are rewarding, that blockade of opioid receptors in either site disrupts a morphine-induced CPP, and that the VTA is more sensitive to both effects.

Animals↗

PubMed1999

Effects of glycine or (+/-)-3-amino-1-hydroxy-2-pyrrolidone microinjections along the rostrocaudal axis of the dorsal periaqueductal gray matter on rats' performance in the elevated plus-maze task.

The effects of glycine (GLY) or (+/-)-3-amino-1-hydroxy-2-pyrrolidone (HA966), a GLY receptor antagonist, microinjections into sites along the rostrocaudal axis of the dorsal periaqueductal gray matter (dPAG) were studied in rats placed on the elevated plus maze (EPM). Selective alterations in the open-arm entries (OAEs) or open-arm time (OAT) of the EPM were the indexes of anxiety. HA966 (30 or 100 nmol, 0.3 microl) increased OAEs and OAT in all 3 dPAG sites, suggesting an anxiolytic effect. GLY (80 and 120 nmol, 0.3 microl) selectively reduced OAEs and OAT, suggesting an anxiogenic effect only when injections were given within the caudal dPAG. When administered together, HA966 reversed the anxiogenic effect of 120 nmol GLY, indicating pharmacological selectivity for the effects of GLY on GLY receptors. Results reinforce an involvement of N-Methyl-D-aspartate-coupled GLY receptors in anxiety and suggest that saturation of this receptor may vary along the dPAG.

Animals↗

PubMed1982

Intracellular transport of microinjected 5S and small nuclear RNAs.

The mechanism by which some RNAs are segregated in the cell nucleus was analysed by microinjecting 32 P-labelled total RNA from HeLa cells into the cytoplasm of Xenopus oocytes. Small nuclear RNAs (u1, U2, U4, U5 and U6) migrated into the cell nucleus, where they became 30-60 fold more concentrated than in the cytoplasm. Other RNAs, such as tRNA and 7S RNA, remained in the cytoplasm, while 5S RNA became concentrated in the nucleolus. Studies with lupus erythematosus antibodies showed that the migrating RNAs become associated with oocyte RNA-binding proteins.

Animals↗

PubMedDate not supplied

Rapid transport of foreign particles microinjected into crab axons.

The rapid transport of optically detectable organelles in axons has been well documented, although its molecular mechanism remains unknown. Here we report that synthetic particles microinjected into the giant axons of the shore crab, Carcinus maenas, are also transported, moving as though they were endogenous organelles. Polystyrene beads, polyacrolein beads, paraffin droplets and glass fragments, of sizes up to 0.5 micron in diameter, have been tested. Many of these foreign particles move rapidly and for long distances along the axon in the anterograde direction, travelling in a saltatory fashion, within a well defined velocity range. In many respects the movements are indistinguishable from those of anterogradely moving endogenous organelles seen by phase-contrast in these axons. Our results indicate that there is a transport system in axons capable of carrying almost any particle of suitable physical properties in an anterograde direction.

Acrolein↗

PubMedDate not supplied

Elevation of tubulin levels by microinjection suppresses new tubulin synthesis.

Most eukaryotic cells rapidly and specifically depress synthesis of alpha- and beta-tubulin polypeptides in response to microtubule inhibitors which cause microtubule depolymerization and presumably increase the intracellular concentration of free subunits. Other drugs which interfere with microtubule function but which lead to a decrease in the subunit pool size have little effect on the rate of new tubulin synthesis. These findings have previously been interpreted to indicate that cultured cells synthesize tubulin constitutively unless the subunit pool rises above a specified level. At this point an autoregulatory control mechanism is triggered which suppresses new tubulin synthesis through specific loss of tubulin mRNAs. That tubulin RNA levels are dramatically lowered by microtubule depolymerizing drugs is unquestionably correct; that fluctuations in the depolymerized tubulin pool size are responsible for altered RNA levels rests, however, entirely on the presumptive effects of different microtubule drugs. This caveat is not trivial, as these drugs induce gross morphological alterations, and the specificities and detailed mechanisms of action of such drugs remain poorly understood. To investigate the effect of altered levels of tubulin subunits on the rate of new tubulin synthesis in mammalian cells, we have microinjected purified tubulin subunits into cells in culture and analysed the synthesized proteins. We report here that tubulin synthesis is rapidly and specifically suppressed by injection of an amount of tubulin roughly equivalent to 25-50% of the amount initially present in the cell, thus indicating the presence of an eukaryotic, autoregulatory control mechanism which specifies tubulin content in a cultured mammalian cell line.

Animals↗

PubMedDate not supplied

Mammalian neural crest cells participate in normal embryonic development on microinjection into post-implantation mouse embryos.

The production of chimaeric mice by aggregating pre-implantation mouse embryos or by injection of cells into the blastocyst has been of great value in analysing the regulation of early mammalian development and in dissecting the relationships of early cell lineages. While the totipotent cells of the pre-implantation embryo can be grown in vitro and thus are readily accessible to experimental manipulation, this is not possible after the embryo has implanted into the uterus. This problem has severely hampered the analysis of cell migration and of cell lineage relationships in later stages of mammalian development. In contrast, the chicken embryo can be manipulated experimentally throughout embryo-genesis and this has made the bird a favourable system for studying patterns of cell migration in the development of higher vertebrates. In mammals, the introduction of retroviruses and haematopoietic cells has provided two means of probing post-implantation development by direct intervention. I report here that cultured neural crest cells, when microinjected into 9-day-old mouse embryos, can migrate over considerable distances and participate in normal development, and the resulting chimaeric animals show pigmentation derived from the donor cells in hair and iris. The introduction of cells into post-implantation embryos may provide the means of studying patterns of cell migration in mammalian development at a level of sophistication which so far has been restricted to the chicken system.

Animals↗

PubMedDate not supplied

Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21.

The protein products (p21) of the ras cellular proto-oncogenes are thought to transduce membrane signals necessary for the induction of cell division. However, there is uncertainty as to the precise role of ras p21 in mediating ligand-membrane receptor signals leading to cell differentiation. Treatment of rat phaeochromocytoma cells (PC12) with nerve growth factor (NGF) results in the induction of a number of phenotypic characteristics of sympathetic neurones, including cessation of cell division and outgrowth of neuronal processes (neurites). Here we report that microinjection of antibody to ras p21 into PC12 cells inhibited neurite formation and resulted in temporary regression of partially extended neurites, an effect which was observed up to 36 h after initiation of NGF treatment. Neurite formation induced by cyclic AMP was unaffected by injection of anti-p21 antibody. These results indicate that p21 is involved in the initiation phase of NGF-induced neurite formation in PC12 cells and has a role in hormone-mediated cellular responses distinct from cell proliferation.

Animals↗

PubMedDate not supplied

Increased phosphorylation of ribosomal protein S6 following microinjection of insulin receptor-kinase into Xenopus oocytes.

The protein products of several transforming retroviruses as well as the receptors for several hormones and growth factors, including insulin, have been shown to possess a protein kinase activity in vitro specific for tyrosine residues in protein substrates, including themselves. In the case of pp60src and the insulin receptor, autophosphorylation activates the tyrosine kinase activity towards exogenous substrates. Experiments indicate that, in vivo, many of these viruses or growth factors induce an increase in cellular phosphotyrosine, as well as an increase in the phosphorylation of serine residues on proteins, including ribosomal protein S6. It seems likely that some of the effects of insulin might be mediated by phosphorylation of intracellular substrates by its receptor. As the beta subunit of the receptor is a transmembrane protein, such phosphorylation could occur either while the receptor is still in the membrane or after its internalization. In various cell systems, internalized receptors are degraded, reshuttled back to the plasmalemma or maintained in a separate compartment before reinsertion in the membrane; shuttling of the insulin receptor could provide the opportunity for it to phosphorylate various intracellular components as part of its mechanism of signal transduction. To approach directly the question of whether the receptor can elicit a signal while acting at an intracellular location, we have microinjected Xenopus oocytes with the insulin receptor kinase. The results indicate that an S6 protein-serine kinase is stimulated or an S6 protein-serine phosphatase inhibited by the activity of the insulin receptor, supporting the concept that the insulin receptor acting within the cell can elicit a biological response.

Animals↗

PubMedDate not supplied

Loss of mouse fibroblast cell response to phorbol esters restored by microinjected protein kinase C.

The phorbol esters in addition to being among the most potent mouse skin tumour promoters profoundly affect many different biological systems. It is postulated that they act through activation of protein kinase C, but substantial heterogeneity in their pharmacological and binding behaviour in some systems has caused concern about whether this is their only target. Evidence linking protein kinase C activation with biological responses to the phorbol esters includes similarity in structure-activity relations for binding and response; in vitro phosphorylation of specific proteins by protein kinase C at the same sites at which phorbol ester treatment induces phosphorylation in intact cells; and correlation in certain cell types between down regulation of protein kinase C on chronic phorbol ester treatment and loss of cellular responsiveness to the phorbol ester. Here we report that microinjection of purified protein kinase C into Swiss 3T3 fibroblasts pretreated with the phorbol ester phorbol 12,13-dibutyrate (PDBu) restores the mitogenic response of the cells to PDBu, directly establishing the involvement of protein kinase C in this response.

Animals↗

PubMedDate not supplied

Release of mature starfish oocytes from interphase arrest by microinjection of human centrosomes.

Mature oocytes (unfertilized eggs) are arrested at definite cell-cycle stages which vary from species to species. In frogs and mammals, the oocytes are arrested at the second metaphase of meiosis whereas in echinoderms they are blocked later, at the pronucleus stage. What causes the maturing oocytes to stop at some point in the cell cycle is not entirely clear. In frogs, the metaphase arrest seems to be maintained by a cytostatic factor. In echinoderms, which stop at interphase, no such a factor has so far been found. The fertilization process, beyond the introduction of paternal chromosomes, releases the oocyte from cell-cycle arrest and provides a functional centrosome to replace the endogenous centrosome which is apparently lost during oogenesis in most species. Several lines of evidence suggest that release from cell-cycle arrest is mediated by a Ca2+ burst which is associated with fertilization, and it is known that the functional centrosome provided by the sperm is necessary for mitotic spindle formation and cleavages. We report here that microinjection of purified human centrosomes into mature starfish oocytes is sufficient to release them from arrest at interphase and to support many cleavages leading to the occasional formation of normal embryos. In this species centrosome induced re-entry into the cell cycle does not require a transient calcium burst nor does it require intact microtubules.

Animals↗

PubMed2002

Inhibition of sperm production in mice by annexin V microinjected into seminiferous tubules: possible etiology of phagocytic clearance of apoptotic spermatogenic cells and male infertility.

Many differentiating spermatogenic cells die by apoptosis during the process of mammalian spermatogenesis. However, very few apoptotic spermatogenic cells are detected by histological examination of the testis, probably due to the rapid elimination of dying cells by phagocytosis. Previous in vitro studies showed that Sertoli cells selectively phagocytose dying spermatogenic cells by recognizing the membrane phospholipid phosphatidylserine (PS), which is exposed to the surface of spermatogenic cells during apoptosis. We examined here whether PS-mediated phagocytosis of apoptotic spermatogenic cells occurs in vivo. For this purpose, the PS-binding protein annexin V was microinjected into the seminiferous tubules of normal live mice, and their testes were examined. The injection of annexin V caused no histological changes in the testis, but significantly increased the number of apoptotic spermatogenic cells as assessed by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. The number of Sertoli cells did not change in the annexin V-injected testes, and annexin V itself did not induce apoptosis in primary cultured spermatogenic cells. These results indicate that annexin V inhibited the phagocytic clearance of apoptotic spermatogenic cells and suggest that PS-mediated phagocytosis of those cells occurs in vivo. Furthermore, the injection of annexin V into the seminiferous tubules brought about a significant reduction in the number of spermatogenic cells and epididymal sperm in anticancer drug-treated mice. This suggests that the elimination of apoptotic spermatogenic cells is required for the production of sperm.

Animals↗

PubMed2006

Precise microinjection into skin using hollow microneedles.

Hollow needles of micron dimensions have previously been fabricated and envisioned for use with transdermal patches or infusion pumps to achieve painless delivery of drugs to the skin for local and systemic effects without the need for hypodermic needles. However, little work has been carried out to identify methods to effectively use hollow microneedles for drug delivery. To address this need, we inserted hollow, glass microneedles into hairless rat skin in vivo and human cadaver skin in vitro and then imaged infusion of dye molecules, insulin, polymer microparticles, and cells into the skin by brightfield and fluorescence microscopy. The depth of needle penetration into skin was controlled by inserting needles with a rotary drilling device, which enabled localized injection within the epidermis or dermis with +/-60 microm resolution. Although small quantities of fluid could be injected after needle insertion into skin, partial retraction of the needle by withdrawing back 100-300 microm or vibrating the microneedle array dramatically increased infusion flow rate. We conclude that hollow microneedles can be used for precise microinjection into skin, especially when a single needle is inserted by rotary drilling and then retracted part way before infusion or a microneedle array is inserted by mechanical vibration.

Cell Transplantation↗